JP2013533308A - Pharmaceutical or cosmetic composition comprising nicotinic acid adenine dinucleotide phosphate or a derivative thereof - Google Patents

Abstract

[PROBLEMS] To provide a composition as a pharmaceutical or functional cosmetic for radically preventing, ameliorating or treating skin diseases and disorders through regeneration and strengthening of a damaged skin barrier, and not merely symptom relief, and use thereof The present invention provides a method for preventing, ameliorating or treating the aforementioned diseases and disorders. Also provided are a composition for promoting differentiation of keratinocytes and a method of promoting differentiation of keratinocytes.
The present invention relates to a use of nicotinic acid adenine dinucleotide phosphate or a derivative thereof for promoting differentiation from a keratinocyte to a fibroblast, and skin barrier regeneration and improvement of a composition containing NAADP or a derivative thereof. The present invention provides a pharmaceutical or cosmetic use for preventing, ameliorating or treating stratum corneum abnormal diseases such as psoriasis and atopic dermatitis, and a use for promoting differentiation of isolated keratinocytes.
[Selection] Figure 2

Description

  The present invention relates to a use of nicotinic acid adenine dinucleotide phosphate (NAADP) or a derivative thereof for promoting differentiation of keratinocytes. More particularly, the present invention relates to skin barrier regeneration and improvement of NAADP or derivatives thereof, pharmaceutical or cosmetic use for prevention, improvement or treatment of stratum corneum abnormal diseases such as psoriasis and atopic dermatitis, and isolation. The present invention relates to a use for promoting differentiation of a formed keratinocyte.

  Human skin is composed of dermis and epidermis. The dermis is mainly composed of fibroblasts that mainly synthesize collagen and other proteins and produce small amounts of lipids. In contrast, the epidermis is mainly composed of keratinocytes that mainly produce lipids and do not substantially synthesize collagen. In particular, the epidermis located at the outermost corner of the skin is protected against various external stimuli such as chemicals, air pollutants, dry environment, physicochemical stimulating factors such as ultraviolet rays and body moisture through the skin. The protection function that prevents excessive divergence is performed. These protective functions are made possible by the normal formation and maintenance of the stratum corneum composed of keratinocytes.

  In the epidermis, the outermost stratum corneum (Stratum corneum, horny layer) is formed from keratinocytes and is composed of differentiated keratinocytes and a lipid layer surrounding them (J. Invest. Dermatol. 1983). 80; 44-49). A stratum corneum is a cell formed through morphological and functional changes in stages while a basal cell that continuously proliferates in the stratum basale moves to the stratum corneum. . After a certain period of time, old keratinocytes fall off the skin, and new keratinocytes rising from the lowermost layer of the epidermis substitute their functions, but this repetitive series of change processes is performed by epidermis differentiation. ) Or keratinization. In this keratinization process, the keratinocytes form a stratum corneum while producing natural moisturizing factor (NMF) and intercellular lipids (ceramide, cholesterol and fatty acid), and the stratum corneum becomes firm and flexible. It has the function as a skin barrier (Skin barrier) that acts as a barrier layer against the outside.

  Such stratum corneum is a habitual element of life such as excessive facial cleansing, taking a bath, environmental factors such as dry air pollutants, and endogenous such as atopic skin and senile skin The function may be easily lost due to diseases. In fact, there are more and more harmful factors to the skin in modern times, and the rate of stratum corneum formation and shedding is slowed by changes in dietary habits. Due to the decreased amount, there is an increasing trend of people with skin whose stratum corneum cannot perform normal skin barrier function.

  Skin dryness, one of the major diseases in modern society, has been shown to be caused mainly by skin barrier dysfunction. Psoriasis is an inflammatory disease of the skin, characterized by epidermal proliferation and skin inflammation, and the skin becomes thick and red due to inflammation, causing scales (keratin). Because it is not contagious but looks bad, patients suffer from stress and poor quality of life. Causes of psoriasis include inhibition of excessive proliferation and differentiation of skin keratinocytes (Roenigk HH, Marcel Dekker Inc, pp. 233-247, 1985), abnormalities of the immune system involving leukocytes such as T cells or cells on resin (JC Prinz, Clinical and Experimental Dermatology, 24, 291-295, 1999) and the like have been proposed. Recently, atopic dermatitis, which occurs in 10% of children, is a chronic re-emergent eczema disease and its pathogenesis is not clear, but chronic skin dryness and the resulting damage to the skin barrier are If it is the main symptom and it is not properly managed, it will progress to lesions such as lichenification, pigment change, and erythroderma. The skin atrophy phenomenon that occurs when a steroid preparation is used for a long time or excessively is a side effect that the skin becomes thin and its function is weakened. The main causes are suppression of fibroblast activity by collagen and collagen. It has been reported that the production is reduced (S. Hammer et al., J. Cell. Biochem, 91, 840-851, 2004).

  Conventionally, a method of increasing moisture retention in the stratum corneum using a humectant that absorbs moisture and an occlusive moisturizer that prevents moisture evaporation has been used. Humectants include substances such as glycerin, propylene glycol, 1,3-butylene glycol, polyethylene glycol, sorbitol, sodium 2-pyrrolidone-5-carboxylate, but have the disadvantage of being very sticky when applied to the skin. There is. As the moisturizing agent, lipid components such as ceramide and essential fatty acids and lipid complexes were used (J. Invest. Dermatol. (5), 731-740, 1994) to maintain the stability of the emulsifier type. However, there are disadvantages that make it difficult to manufacture a transparent zel-like product. In addition, when using the above-described moisturizing agent having a normal water retention function, temporary relief can be expected, but radical healing is difficult. Therefore, there is a rapid development of substances that promote differentiation of keratinocytes and promote fibroblast growth to fundamentally regenerate damaged barriers.

  On the other hand, recently, it has been proposed that stratum corneum differentiation is important to prevent skin moisture from drying, and the differentiation process developed from the stratum corneum of the basal layer to the stratum corneum of the outermost stratum corneum The fact that it can play a role as a skin moisture retention barrier only after it has been done successfully has become clear. That is, in the process of keratinization, cells generate natural moisturizing factor (NMF) and intercellular lipids, and as a result, the stratum corneum becomes rigid and flexible and functions as a defense wall. It can be done. From the physiological viewpoint of the tendency of the skin to dry as the age of humans increases, the amount of moisturizing factor and lipid in the stratum corneum increases due to a longer stratum corneum shedding time or decreased lipid synthesis ability of epidermal cells. Can be analyzed. Thus, by promoting the differentiation of keratinocytes, a new approach is possible that can induce strengthening of the skin barrier and enhance the skin moisture maintenance function and the protective function from the external environment.

Korean Published Patent No. 10-2009-0120350

  The object of the present invention is not merely symptom relief, but as a pharmaceutical or functional cosmetic composition for fundamentally preventing, ameliorating or treating skin diseases and disorders through regeneration and strengthening of damaged skin barriers and It is to provide a method for preventing, ameliorating or treating the diseases and disorders using the same. Another object of the present invention is to provide a composition for promoting differentiation of keratinocytes and a method of promoting differentiation of keratinocytes.

In order to achieve the above-mentioned object, the present invention provides a composition comprising a nicotinic acid adenine dinucleotide phosphate (NAADP) or a derivative thereof represented by Chemical Formula 1 as an active ingredient and a method of using the same. provide.
Where
R ₁ and R ₂ are each independently H, C _1-4 alkyl (which can be unsubstituted or substituted with halogen), or CH ₂ —CO—CH ₃ ;
W is selected from the group consisting of NH ₂ , OH and SH;
X is selected from the group consisting of OH, SH, NH ₂ and halogen; and Y is selected from the group consisting of OH, H, NH ₂ and halogen.

  NAADP used in the present invention is synthesized in cells by ADP-ribosyl cyclase containing CD38 (Chini EN. Et al., Biochem J 362: 125-130, 2002; BERRIDGE G. et al. Biochem. J., 365: 295-301, 2002; Aarhus R. et al., J Biol Chem., 270 (51): 30327-30333, 1995), the intracellular calcium concentration of all living organisms. It is important for the maintenance of the life phenomenon of cells as a substance that is used to regulate.

  The present inventors are the first in the world that NAADP has a function of improving the differentiation ability of keratinocytes in the process of processing NAADP into human keratinocytes and conducting research related to differentiation. discovered. Specifically, the present inventors have promoted the release of calcium in cells as NAADP as a calcium signal regulatory molecule in keratinocytes (FIG. 1), and the involucrin, keratin 1, and keratin, which are differentiation indicators of keratinocytes. It was clarified that it has the effect of increasing the expression of 10 (FIG. 2). Furthermore, the present inventors completed the present invention by producing a dosage form of an external preparation containing NAADP and applying it to the skin of animals and humans to obtain a result of strengthening the skin barrier (FIG. 3). .

The present invention will be described in detail below.
The present invention provides a composition that promotes differentiation of keratinocytes, comprising NAADP represented by Chemical Formula 1 or a derivative thereof as an active ingredient.
In addition, the present invention provides a composition for regenerating and reinforcing a skin barrier comprising NAADP represented by Chemical Formula 1 or a derivative thereof as an active ingredient.

  The composition comprising NAADP or a derivative thereof represented by Chemical Formula 1 of the present invention as an active ingredient regenerates and strengthens the skin barrier by promoting differentiation of keratinocytes, and chronic such as psoriasis, atopic dermatitis, ichthyosis, etc. Skin barrier damage disease, primary contact dermatitis, contact dermatitis like allergic contact dermatitis, acute skin barrier damage disease like allergic dermatitis, dry skin, eczema, skin atrophy due to steroid side effects, skin It can be effectively used for the prevention or treatment of skin diseases and disorders such as, but not limited to, wounds, scars, wrinkles, skin aging, occurrence of melasma, weak skin elasticity. In the above, dry skin is not limited to skin aging, environmental causes, but also to other causative diseases such as diabetes, myxedema, lymphoma, tumor, acquired immune deficiency syndrome, hereditary diseases such as Down syndrome Including cases where it is caused and zinc deficiency, dryness due to diuretics or antihistamines. The scars on the damaged skin remain worse as it gets older, due to a decline in the ability of the skin to regenerate. Since the reduction of skin regeneration ability can delay recovery of wounds and deepen the reduction of skin elasticity, the composition of the present invention can increase skin regeneration ability to improve these problems. The stratum corneum contains a natural moisturizing factor (NMF) that plays an important role in moisturizing the skin, and this natural moisturizing factor is produced in the final differentiation process of keratinocytes. Are known. Therefore, the composition containing NAADP or a derivative thereof represented by the chemical formula 1 of the present invention as an active ingredient promotes the production of NMF in addition to the mechanism for regenerating the skin barrier. It can also function as a humectant.

  NAADP or a derivative thereof used in the present invention is not only a free substance, but also a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, a pharmaceutically acceptable polymorph, or a pharmaceutically acceptable It can be understood that it can be provided as an acceptable precursor agent. The active ingredient may be used alone or in the form of a conjugate or aggregate with other pharmaceutically active compounds.

  In the salt of the compound by this invention, if it is a form which can be mix | blended among pharmaceuticals or cosmetics, it will not restrict | limit in particular. An inorganic salt or an organic salt is included, and an acidic salt or an alkaline salt may be used. In particular, in the case of a salt with a cation, an alkali metal salt such as sodium salt or potassium salt; an alkaline earth metal salt such as calcium salt, magnesium salt or barium salt; a basic amino acid salt such as arginine or lysine; Ammonium salts such as cyclohexylammonium salt; various alkanolamine salts such as monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, diisopropanolamine salt and triisopropanolamine may be used. Preferably, the salt is an alkali metal salt, more preferably a tetrasodium salt.

  The present invention provides a pharmaceutical composition that promotes differentiation of keratinocytes, comprising NAADP represented by Chemical Formula 1 or a derivative thereof as an active ingredient. Alternatively, the present invention provides a pharmaceutical composition for regenerating and strengthening skin barrier, comprising NAADP represented by Formula 1 or a derivative thereof as an active ingredient. In addition, this invention provides the pharmaceutical formulation for skin disorder | damage | failure and disease treatment, especially skin external preparation containing the effective amount of the said composition.

  The present invention also provides a method for preventing, treating or ameliorating the above skin disorders and diseases by administering the composition or formulation to a mammal.

  The pharmaceutical composition containing the NAADP of the present invention or a derivative thereof may further contain appropriate carriers, excipients and diluents commonly used in the production of pharmaceutical compositions.

  When formulating, it is manufactured using excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are usually used. Anti-flocculating agents, lubricants, fragrances, sulfiding agents, preservatives, and the like may further be included in the industry to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Dosage forms may be made using well known methods.

  A pharmaceutical composition comprising NAADP or a derivative thereof according to the present invention may be formulated into conventional pharmaceutical dosage forms known in the technical field to which the present invention belongs. The dosage forms include topical preparations (topical application, patching and iontophoresis), oral administration preparations, injections, suppositories, transdermal administration preparations, transdermal administration preparations and inhalation administration preparations. Although it may be formulated into any dosage form and administered, it may be preferably formulated into a transdermal preparation and a topical skin preparation for topical application.

  In one embodiment, the composition of the present invention is an external preparation for skin, which is a solution, suspension, emulsifier, ointment, paste, gel, cream, lotion, powder, spray, pack, patch for skin adhesion, dressing, It can be formulated into all dosage forms applicable to the skin, such as hydrous or non-hydrous attachments.

  When the dosage form of the present invention is a paste, cream or gel, the carrier component includes animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. It may be used.

  When the dosage form of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. , Propellants such as propane / butane or dimethyl ether may be included.

  * When the dosage form of the present invention is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, There are fatty acid esters of benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

  When the dosage form of the present invention is a suspension, the carrier component is a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester. Suspensions, microcrystalline cellulose, aluminum metahydroxide, bentonite, aga or tragacanth may be utilized.

  In the external preparation for skin of the present invention, cosmetics applied to the skin and mucous membranes, and various known ingredients to be added to external medicines and quasi drugs can be mixed in application amounts.

  Such ingredients include antioxidants (eg, carboxylic acids such as ascorbic acid, citric acid; phenols such as tocopherol, dibutylhydroxytoluene), preservatives (eg, dehydroacetic acid, salicylic acid, edetate disodium Carboxylic acids such as: ethyl paraoxybenzoate, methyl paraoxybenzoate, isopropyl paraoxybenzoate, phenols such as thymol), wetting agents (eg glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol) Glycols; organic salts such as hyaluronic acid; amides such as iodine], thickeners (eg, polymer compounds such as polyethylene glycol; cellulose such as sodium carboxymethylcellulose and carboxypropylcellulose) ), Buffers (for example, organic acids such as citric acid, lactic acid and tartaric acid; inorganic acids such as hydrochloric acid and boric acid; salts such as sodium dihydrogen phosphate and sodium citrate; and triethanolamine Organic salts; inorganic bases such as marine product sodium and marine product potassium), adsorbents (eg, hydrous aluminum silicates such as kaolin and bentonite; inorganic salts such as marine product magnesium magnesium and marine product aluminum), Base (eg white petrolatum, Tween 60, Tween 80, liquid paraffin, beeswax, petrolatum, castor oil, silicone oil, hydrogenated castor oil, natural rubber, palm oil fatty acid diethanolamide, polyoxyethylene hydrogenated castor oil, natural rubber latex, 1,3 -Organic substances such as pentadiene copolymer resin; polybutene, synthesis SBR, polyethylene glycol monostearate, polyoxyglycol monostearate, polyoxyethylene cetostearyl ether, polyoxyethylene oleyl cetyl ether, silicone, starch acrylate 300, sodium polyacrylate, methacrylic acid / acrylic acid n- High molecular compounds such as butyl copolymer and carboxyvinyl polymer; fatty acids such as stearic acid; alcohols such as cetanol and myristyl alcohol; fatty acid esters such as octadodecyl myristate, isopropyl myristate and cetyl octanoate) , Carbohydrates such as solvents (eg, ethanol, isopropanol, 1,3-butylene glycol, n-octadecyl alcohol, crotamiton, tri (caprylic acid / capron) glycerin) ), Stabilizers (eg, inorganic salts such as sodium metaphosphate, zinc oxide, titanium oxide; organic salts such as sodium polyoxyethylene lauryl sulfate ether sulfate and sodium lauryl sulfate), adhesives (eg, polyacrylic acid) High molecular weight compounds such as sodium and dipropylene glycol), emulsifiers (eg, sorbitan monoolefin acid, polyoxyethyl sorbitan monoolefin acid, D-sorbitol, polyglycerin monolaurate, carbohydrates such as sodium polyoxyethylene lauryl ether sulfate) ), Surfactants (for example, high molecular weight compounds such as polyglycerol monolaurate and polyoxyethylene oleyl alcohol ether), perfumes, dyes (dyes, pigments), metal sequestering agents, deodorants, film forming agents , UV absorber Ultraviolet light scattering agent, and the like vitamins.

  The composition used as an external preparation for skin additionally contains a substance for promoting skin absorption in order to increase the effect of enhancing the skin barrier function, the effect of inducing differentiation of skin keratinocytes and the effect of moisturizing the skin. May be.

  Moreover, in manufacturing the external skin solvent of this invention, unless the pharmacological effect is harmed, another medicinal component may be included. This medicinal component is, for example, a known refreshing component, cortical inhibitor, antibacterial agent, bactericidal agent, anti-inflammatory agent, skin astringent, cell rejuvenating agent, vasodilator, performance promoting agent, skin function antipromoting agent and herbal medicine is there. Specifically, menthol, salicylic acid, estradiol, glycyrrhizic acid, benzalkonium chloride, phenol, camphor, etc .; narcotics and stimulants such as ethylmorphine hydrochloride, oxycodone hydrochloride, cocaine hydrochloride, pethidine hydrochloride, methamphetamine hydrochloride, dl-methylephedrine hydrochloride , Morphine hydrochloride, fentanyl citrate, levalol bread tartrate, etc .; topical bactericides such as fobidone iodine, iodoflum, etc .; enzyme preparations such as lysozyme chloride, streptokinase, streptodornase trypsin, deoxyribonuclease, etc .; , Siconex, lotex, etc.

  Aqueous suspensions are generally one unusual suspending agent such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragagant and gum acacia; dispersants or wetting agents such as lecithin or oxidized A condensation product of an alkylene and a fatty acid (eg, polyoxylene stearate), or a condensation product of a long chain aliphatic alcohol such as ethylene oxide and heptadecaethyleneoxycetanol, or a partial ester derived from a fatty acid and hexitol Condensation products of ethylene oxide, for example polyoxyethylene sorbitol monooleate, or condensation products of long chain aliphatic alcohols such as ethylene oxide and heptadecaethylene oxetanol, or fatty acids and Condensation products of partial esters derived from sitol and ethylene oxide, such as polyoxyethylene sorbitol monooleate, or condensation products of partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan mono Contains active ingredient in fine powder form with oleate. Aqueous suspensions may contain one or more other preservatives (eg ethyl or propyl p-hydroxybenzoate, antioxidants (eg ascorbic acid), colorants, fragrances and / or sweeteners (eg sucrose, saccharin or Saccharin or aspartame).

  Oily suspensions may be formulated in suspension with the active ingredient in vegetable oil (eg, peanut oil, olive oil, sesame oil or coconut oil) or mineral oil (eg, liquid paraffin). The oily suspension may also contain a demulcent such as beeswax, hard paraffin or cetyl alcohol. A sweetening agent and a fragrance as described above can be added to provide a palatable oral preparation. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

  Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavoring and coloring agents may also be present.

  The pharmaceutical composition of the present invention may also exist in the form of an oil-in-water emulsion. The oil may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture of any of these. Suitable emulsifiers are, for example, natural-generated gums such as gum acacia or gum tragacanth, natural-generated phosphatides such as soy, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides (eg sorbitan monooleate) And a condensation product of the partial ester and ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening, flavoring and preserving agents.

  Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain demulcents, preservatives, fragrances and / or coloring agents. Also good.

The appropriate dosage and timing of the composition of the present invention vary depending on the formulation method, age, sex, body weight, degree of disease symptoms, excretion rate and response sensitivity, administration route and period of the subject of administration. A dose effective for the intended treatment can be appropriately selected. For example, in an orally-administered preparation, it can be administered in a predetermined number of times so as to be 0.01 to 10.0 g / kg, preferably 0.1 to 1.0 g / kg per day based on adults. . In the preparation for external use on the skin, the effective amount is in the range of 1 nM to 10 mM, preferably 1 nM to 100 μM per day of the skin surface area (cm ² ), and can be applied once to 5 times a day. The dosage form of the composition may be a single dosage or a repeated dosage form, and the daily effective dose may be administered in a predetermined number of times.

  The pharmaceutical composition of the present invention can be used as a single therapy, but may be used in combination with surgery, hormone therapy, radiation therapy, drug therapy, methods using biological response modifiers, diet therapy, and the like. Good.

  The composition of the present invention may further include cells constituting the skin, such as keratinocytes, in addition to NAADP of Formula 1 or a derivative thereof.

  In a further aspect of the present invention, the present invention provides a functional cosmetic composition for improving skin barrier function and moisturizing skin, comprising NAADP of Formula 1 or a derivative thereof. The composition of the present invention enhances the protective function from the external environment by promoting the differentiation of keratinocytes and restoring the skin barrier function, so that skin wounds, scars, wrinkles, reduced elasticity or atopic dermatitis To prevent or ameliorate skin disorders or diseases such as eczema, or psoriasis. Moreover, it has the effect of improving the skin moisture maintenance function, releasing the tension and roughness of the skin and increasing the gloss.

  The functional cosmetic containing the NAADP of the present invention or a derivative thereof as an active ingredient can be applied in all dosage forms applied to the skin. More specifically, not only cosmetics such as skins, lotions, gels, creams, essences, water-soluble powders, fat-soluble powders, water-soluble liquids, foundations, sprays, packs, cleansing foams, cleansing creams, body lotions, It can be made into body creams, body oils, body cleansers, soaps, shampoos, ointments and patches. In addition, it can be manufactured as a skin contact material that comes into contact with the skin such as makeup, detergent, and fiber.

  In the cosmetic composition of each dosage form, other components besides the NAADP or its derivative can be appropriately selected and blended by those skilled in the art within a range not damaging the object and effect of the present invention. . The ingredients that can be added include oil and fat ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, and pH compositions. Alcohol, pigment, fragrance, blood circulation promoter, cooling agent, antiperspirant, purified water, and the like.

  In addition to NAADP or a derivative thereof, the cosmetic of the present invention may further include a component washed from the group consisting of water-soluble vitamins, fat-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and seaweed extracts. Good.

  The cosmetic composition of the present invention can be applied by applying an appropriate amount to the skin according to the skin area to be applied, and used once to several times a day as necessary. Can do. The amount and frequency of application can be increased or decreased as necessary according to the skin condition, age, etc. of the individual.

  In another aspect of the invention, the invention comprises contacting the composition with a skin stratum corneum or a culture of keratinocytes in vivo, ex vivo, or in vitro. A method of promoting differentiation of keratinocytes is provided.

  Cultures of keratinocytes are not only widely used for the treatment of skin damage such as burns, chronic diseases, leukoplakia, but are also used to study the differentiation steps of cells. In order for keratinocytes to be applied medically, it is necessary to proliferate in large quantities in a culture vessel from a small amount of cells obtained from the human body.

  In one embodiment, the method comprises contacting a keratinocyte isolated from a mammal or a keratinocyte cultivated separately with a composition comprising NAADP of Formula 1 or a derivative thereof. Differentiated cells obtained by the above method may be used in in vivo or in vitro experiments, and can be used for transplantation injections and transplants for tissue restoration / regeneration, artificial skin, etc. Is not. Said method may further comprise the step of applying an effective amount of the composition according to the invention to the skin of a mammalian individual in need of epidermal regeneration and differentiation. The effective amount of the composition, the number of applications, the interval and the period can be appropriately selected by those skilled in the art according to the purpose.

  The present invention also provides a kit containing NAADP of Formula 1 or a derivative thereof, which is used for the purpose of promoting differentiation of keratinocytes.

  In yet another aspect, the present invention provides a method of using the composition for the manufacture of a medicament for the treatment of the skin diseases and disorders.

In another aspect, the present invention provides a method for screening a substance capable of regulating the keratinocyte differentiation ability of a test substance using the composition of the present invention. The method
Providing isolated human keratinocytes;
Contacting the isolated human keratinocytes with a composition containing NAADP or a derivative thereof, and each test substance;
Detecting the amount of calcium increase or differentiation indicator expression in the keratinocytes;
Determining the ability of the test substance to induce differentiation of keratinocytes by comparing the amount of calcium increase or the expression level of differentiation indicator detected from the keratinocytes in contact with the composition and the test substance.

  Human keratinocytes used in such screening methods may be any cells as long as they can be carefully discriminated from changes in calcium and differentiation indicator expression levels by culturing each in the presence of the composition of the present invention and the test substance. It is possible even if it exists. Preferably, a cultured cell line consisting of normal cells derived from human foreskin and human keratinocytes (Federer keratinocyte neonate, NHEK-Np, CC-2507, Lonza Co., Ltd.) is appropriately used as a feeder layer. can do.

  The differentiation indicator of keratinocytes may be a specific protein such as involucrin, loricrin, keratin 1 or keratin 10, but the present invention is not limited to this, and the presence / absence and amount of expression are as in Western block. It can be measured using conventional protein detection methods.

  The amount of calcium increase or the expression level of the differentiation indicator can be evaluated by a known measurement method to evaluate the ability of the test substance to induce differentiation of keratinocytes and the degree of efficacy to regenerate and strengthen the skin barrier. .

  The composition comprising NAADP or a derivative thereof of the present invention exhibits an effect of increasing the skin moisturizing power by restoring damaged skin barrier function through promoting differentiation of keratinocytes. Therefore, the composition containing NAADP or a derivative thereof may be used as a pharmaceutical composition or functional cosmetic composition for skin barrier regeneration and strengthening and skin moisturization, and induces differentiation of keratinocytes in vitro. It may be used as a substance. In addition, the pharmaceutical or cosmetic containing the composition may be used for psoriatic disease, atopic dermatitis, chronic skin barrier damage diseases such as ichthyosis, primary contact dermatitis, contact dermatitis such as allergic contact dermatitis, allergy Skin diseases and disorders such as acute skin barrier damage diseases such as dermatitis, dry skin, eczema, skin atrophy due to side effects of steroids, skin wounds, scars, wrinkles, skin aging, occurrence of melasma, weakened skin elasticity It can be used effectively for prevention or treatment.

It is the graph which showed the influence which acts on the calcium density | concentration change in the keratinocyte which the NAADP containing primary culture | cultivated primarily classified. It is the western block photograph which evaluated the influence which the composition containing NAADP exerts on the differentiation of the morphogenic cells primarily cultured using the expression levels of differentiation indicators, involucrin, keratin 1 and keratin 10. It is a photograph of a mouse skin tissue showing the degree of differentiation induction of the skin stratum corneum depending on the concentration when a composition containing NAADP is applied to a hairless mouse.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, these examples are merely examples for helping understanding of the present invention, and the scope of the present invention is not limited thereby.

Example 1: Verification of the effect of NAADP on the increase in calcium in keratinocytes Normally, the epidermis has a calcium gradient, and the concentration of calcium ions is lower in the basal layer and the spinous layer, and the higher the layer is, the higher the layer is. Increased highest in outer grain layer. Damage to the skin barrier induces water loss from the top of the skin, which results in changes in the calcium gradient within the epidermis. Such a change in calcium gradient induces a homeostatic response in the skin whereby the skin barrier function is quickly restored to continue to form a normal form of calcium gradient. That is, it became clear that changes in calcium ions play an important role as a signal to initiate the recovery process after skin barrier damage.
In this example, in order to confirm the recovery effect of the skin barrier in the composition of the present invention, when NAADP was treated to cultured keratinocytes, the effect of inducing intracellular calcium increase was evaluated.

Method : Calcium fluo-3 AM (Molecular Probes, USA) in cells is added to keratinocytes (Epidmal Keratinocyte neonate, NHEK-Np, CC-2507, Lonza Co. LTd.) In a CO ₂ incubator for 30 minutes. After the treatment, it was washed 3 times with KBM medium (Keratinocyte Basal Media, KBM-2, Lonza Co. Ltd.). Intracellular calcium was measured from the KBM medium, and an increase in intracellular calcium due to NAADP was observed. Intracellular calcium was measured using a confocal microscope (Confocal microscope, Nikon, Japan) equipped with a laser light source having an excitation wavelength of 488 nm and an emission wavelength of 530 nm to collect calcium images. Calculate the concentration. FIG. 1 shows the amount of calcium change measured when keratinocytes (5 × 10 ⁵ cells) were treated with NAADP at 0.1, 1, 10, 100, and 1000 nM.

Results When keratinocytes were treated with 1 to 100 nM NAADP, it was confirmed that calcium movement in the cells was induced. Based on the fact that calcium acts as an important signaling substance in the damaged skin barrier, it was confirmed that the composition containing NAADP according to the present invention exhibits a quick recovery effect of the skin barrier function. This result suggests that it is effective in the treatment of wounds, atopic dermatitis and psoriasis, eczema, skin aging prevention, etc. together with the results of induction of differentiation of keratinocytes as described below.

Example 2: Verification of the differentiation promoting effect of NAADP on keratinocytes The keratinocytes are the cells that form the outermost horny layer of the skin and play a very important role in the moisturizing and protective functions of the skin, and are excessively proliferated. Are preferably suppressed, cell death is suppressed, and differentiation is preferably promoted. Excessive proliferation causes the skin stratum corneum to become abnormally thick and causes the skin to become rough and thick, and abnormal differentiation prevents normal skin barrier functioning, so dry skin and atopic dermatitis Increase the chances of causing several problems, such as psoriasis.
During differentiation of keratinocytes, the expression of regenerated differentiation indicators (involucrin, keratin 1 and keratin 10) was measured using the Western block method to test the effect of promoting NAADP cell differentiation.

Method Human keratinocytes that were primarily cultured were placed in a culture flask and allowed to adhere to the bottom. NAADP diluted according to concentration and retinoic acid as a control group were added to the culture solution, so that the cells had a bottom area of 70 to 80. The cells were cultured for 5 days until they reached about%. The cells were harvested and washed with phosphate buffered saline (PBS). Subsequently, 1 ml of a tris-HCl buffer solution (Tris-HCl, pH 7.4) containing 10% concentration of 2% sodium dodecyl sulfate (SOD) and 20 mM dithiothreitol (Dithiothreitol, DTT). Add the sonicated, boiled and centrifuged precipitates to 1 ml of PDS. The protein pigment in the suspension was measured and used as a reference when evaluating the degree of cell differentiation. FIG. 2 shows the results of a test carried out with keratinocytes (5 × 10 ⁵ cells) treated with retinoic acid (1 μM) treated as a positive control group and different concentrations of NAADP (10 pM to 10 μM) added to the low calcium concentration. It was.

Results As a result of evaluating the effect on the differentiation of keratinocytes, it was confirmed that the differentiation indicator was expressed from the NAADP treatment group having a concentration of 10 pM to 10 μM as shown in FIG. 2, and keratin 1 and keratin 10 showed a better effect. Therefore, it was confirmed that the substance described above has an effect of improving the differentiation ability of keratinocytes. It has now been found that the compounds according to the invention can promote the differentiation of keratinocytes and consequently rapidly restore the skin barrier function.

Example 3: Verification of differentiation induction effect of NAADP skin stratum corneum in hairless mice Hairless mice (SPF / VAF Crl: SKH1-hr) used in this study were purchased from Orient Bio (Sonnan, Korea). The NAADP of the experimental material of the present invention was a product of Sigma.

Method A test substance is prepared by dissolving NAADP according to concentration in a solution obtained by adding 1% DMSO to a phosphate buffer solution, and this is applied to the right and left backs of hairless mice (Hairless mouse) around 6 weeks with a cotton swab. The test was conducted at Each concentration was applied 5 times, and was performed twice a day at 9 am and 7 pm. Animals with drug applied for 1 week were sacrificed by deboning of the cervical spine, skin was incised 1 × 1 cm and fixed overnight with 10% formalin solution. The fixed tissue was stained with an antibody (Santa Cruz # SC53251) that recognizes keratins 1 and 10 at a 1: 100 dilution factor by preparing a paraffin block and a 5 μm section. As a secondary antibody, anti-mouse IgG (invitrogen) to which Alexa488 was bound was used.
The control group used a solution obtained by adding 1% DMSO to a phosphate buffer solution, and the test group used solutions prepared by diluting NAADP to various concentrations in the solution used for the control group.

As a result, the NAADP-treated group can confirm that the expression levels of keratin 1 and keratin 10 increase in the outer skin part of the skin compared to the control group, and the concentration indicated by the increased expression level of the relevant differentiation indicator is 100 nM or more. You can also see that.
In vitro studies have shown that high concentration NAADP treatment reduces calcium increase and differentiation indicator expression (see Examples 1 and 2), but in this experiment keratin depends on NAADP concentration. 1 and keratin 10 were confirmed to increase. This is considered to be due to the difference between the conditions using only animal cells and the process absorbed through the skin barrier in vivo.

Formulation example
Formulation Example 1 Cream for external use (100g)
A humectant and NAADP were added to purified water and heated to 70 ° C. to adjust. The milk components were dissolved by heating, and adjusted to 70 ° C. by adding an emulsifier, a preservative and the like. This was added to the previous water state, and the emulsified particles were homogenized with a homomixer, and then degassed, filtered and cooled.
Main ingredient NAADP 1.0mg
Milk ingredient Setostearyl alcohol 6.0g
Stearic acid 2.0g
Lanolin 4.0g
Squalane 9.0g
Octyldodecanol 10.0g
Moisturizer 1,3-butylene glycol 3.0g
Glycerin 2.0g
Emulsifier POE (25) cetyl alcohol ether 3.0g
2.0g glyceryl monostearate
* Preservative Propylparaben appropriate amount Methylparaben appropriate amount Purified water remaining

Formulation Example 2 External lotion (100g)
NAADP and a humectant were added to purified water and heated to 70 ° C. for adjustment. The milk components were dissolved by heating, and adjusted to 70 ° C. by adding an emulsifier, a preservative and the like. After adding this to the previous water state and emulsifying with a homomixer, hyaluronic acid 1 aqueous solution was added and homogeneously mixed with the homomixer, and then degassed, filtered and cooled.
Main ingredient NAADP 1.0mg
Milk component cetostearyl alcohol 1.0g
Beeswax 0.5g
Petrolatum 2.0g
Squalane 6.0g
Dimethyllolysiloxy acid 2.0g
Emulsifier POE (10) oleate 1.0g
Glycerol monostearate 1.0g
Moisturizer Glycerin 4.0g
1,3-butylene glycol 4.0 g
Preservative Propylparaben Appropriate amount Methylparaben Appropriate amount Purified water Remaining

Formulation Example 3 Gel (100g)
Polyethylene glycol is added and dissolved in purified water, and NAADP is added and dissolved by heating. This is cooled to about 50 ° C., and added with stirring and added to polypropylene glycol and glycerin with polyoxyethylene cetyl ether and heated to about 50 ° C. Also, with continuous agitation, add aquatic sodium and prepare the pH to about 6.8. After cooling to about 40 ° C., isopropanol is added and cooled to about 25 ° C., then collected in a suitable container.
NAADP 1mg
Polyethylene glycol 8g
Carboxy vinyl polymer 0.5g
Methylcellulose 0.2g
5g propylene glycol
Glycerin 2g
Polyoxyethylene oleyl cetyl ether 1g
Isopropanol 5g
Sodium hydroxide appropriate amount Purified water remaining

Formulation Example 4 Nutrition cream (100g)
Prepared according to the method of Formulation Example 1 using the following ingredients.
NAADP 0.5mg
Stearyl alcohol 6.0g
Stearic acid 2.0g
Concentrated glycerin 1.0 g
Squalane 9.0g
1,3-butylene glycol 6.0 g
Polysorbate 60 1.5g
Polyethylene glycol 1000 4.0g
Hardened lanolin 4.0g
Octyldodecanol 10.0g
Sorbitan stearate 0.8g
Triethanolamine 0.5g
Preservative Appropriate amount Pigment Appropriate amount Purified water Remaining

Claims (10)

Shown below in Chemical Formula 1:
Where
R ₁ and R ₂ are each independently H, C _1-4 alkyl (which can be unsubstituted or substituted with halogen), or CH ₂ —CO—CH ₃ ;
W is selected from the group consisting of NH ₂ , OH and SH;
X is selected from the group consisting of H, OH, SH, NH ₂ and halogen; and Y is selected from the group consisting of OH, H, NH ₂ and halogen, nicotinic acid adenine dinucleotide phosphate (Nicotinic acid adenine dinucleotide phosphate, NAADP) or a derivative thereof, or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate or pharmaceutically acceptable polymorph thereof as an active ingredient A pharmaceutical composition for regenerating or strengthening.   Psoriasis, atopic dermatitis, ichthyosis, primary contact dermatitis, allergic contact dermatitis, allergic dermatitis, dry skin, eczema, skin atrophy due to steroid side effects, skin wounds, scars, wrinkles, skin aging, The pharmaceutical composition according to claim 1, which is used for the prevention or treatment of skin diseases and disorders such as occurrence of melasma and weakened skin elasticity.   Selected from the group consisting of solutions, suspensions, emulsifiers, ointments, gels, pastes, creams, lotions, powders, sprays, packs and patches for skin adhesion, dressings, hydrous or non-hydrous attachments The pharmaceutical composition according to claim 1 or 2, which is formulated into a skin external preparation. 4. The pharmaceutical composition according to claim 3, wherein the dosage of the active ingredient is 1 nM to 100 μM per day (cm ² ) in the case of an external skin solvent. Shown below in Chemical Formula 1:
Where
R ₁ and R ₂ are each independently H, C _1-4 alkyl (which can be unsubstituted or substituted with halogen), or CH ₂ —CO—CH ₃ ;
W is selected from the group consisting of NH ₂ , OH and SH;
X is selected from the group consisting of OH, SH, NH ₂ and halogen; and Y is selected from the group consisting of OH, H, NH ₂ and halogen, nicotinic acid adenine dinucleotide phosphate (NAADP) Or a derivative thereof, or a functional cosmetic composition for regenerating, reinforcing or moisturizing the skin barrier, comprising as an active ingredient a salt, solvate or polymorph thereof.   Psoriasis, atopic dermatitis, ichthyosis, primary contact dermatitis, allergic contact dermatitis, allergic dermatitis, dry skin, eczema, skin atrophy due to steroid side effects, skin wounds, scars, wrinkles, skin aging, The functional cosmetic composition according to claim 5, which is used for generation of melasma, skin elasticity weakening, skin tension, rough or dull skin, prevention or improvement.   The composition includes skin, lotion, gel, cream, essence, water-soluble powder, fat-soluble powder, water-soluble liquid, foundation, spray, cosmetics including pack, cleansing foam, cleansing cream, body lotion, body cream, body The functional cosmetic composition according to claim 5 or 6, wherein the functional cosmetic composition is manufactured in a form selected from the group consisting of oil, body cleanser, soap, shampoo, ointment and patch. Shown below in Chemical Formula 1:
Where
R ₁ and R ₂ are each independently H, C _1-4 alkyl (which can be unsubstituted or substituted with halogen), or CH ₂ —CO—CH ₃ ;
W is selected from the group consisting of NH ₂ , OH and SH;
X is selected from the group consisting of OH, SH, NH ₂ and halogen; and Y is selected from the group consisting of OH, H, NH ₂ and halogen, nicotinic acid adenine dinucleotide phosphate (NAADP) ) Or a derivative thereof, or a salt, solvate or polymorph thereof as an active ingredient, a composition for promoting differentiation of skin keratinocytes.   Promoting the differentiation of mammalian keratinocytes, comprising contacting the composition of claim 8 with keratinocytes isolated from mammalian skin or keratinocytes isolated and cultured from skin. Method. A kit for promoting differentiation of keratinocytes, comprising the composition according to claim 8.